Project Summary NF-?B is a family of transcription factors that are expressed in all mammalian cell types and act as the master regulator of the immune response. Aberrant activation of NF-?B has been implicated in many inflammatory and autoimmune diseases, as well as cancer. NF-?B can be activated by two different signaling pathways, the canonical (or classical) pathway and the non-canonical pathway. Canonical NF-?B signaling involves a key interaction between inhibitor of ?B (I?B)-kinase ?/? (IKK?/?) and NEMO (also called IKK?). Inhibition of the NEMO-IKK?/? interaction thus represents an attractive anti-inflammatory and anticancer strategy, as the inhibitor would block the NF-?B activity induced by pro-inflammatory stimuli but not the basal NF-?B activity, which is required for normal B- and T-cell function. Unfortunately, previous attempts to develop small-molecule inhibitors against this intracellular protein-protein interaction have been largely unsuccessful. In this project, we will explore cell-permeable macrocyclic peptides for inhibition of the NEMO-IKK?/? interaction. Specific Aim 1 is to further optimize the potency, selectivity, and other drug properties of a bicyclic peptidyl inhibitor (NI174-5; IC50 = 1.0 M) discovered during our preliminary studies and biochemically, biologically, and structurally (by X-ray crystallography and in silico modeling) characterize the improved NEMO inhibitor(s). Specific Aim 2 is to determine the effect of NI174-5 (or an improved NEMO inhibitor) on ovarian cancer stem cells (CSC) in vitro and in vivo, as well as its ability to prevent tumor relapse following initial cisplatin treatment in an orthotopic ovarian tumor xenograft model.